PROJECT SUMMARY Elucidation of the pathways and factors that mediate microRNA (miRNA) repression of mRNAs is critical to understanding the biology of these ubiquitous small RNAs and utilizing them in applied and therapeutic settings. miRNAs mediate post-transcriptional repression of mRNAs by binding the 3? UTR of mRNAs through base-pairing interactions in the context of an Argonaute (AGO) family protein. Despite progress in identifying features of target sites that are correlated with observed repression, there exists a major gap in the field's understanding of how miRNAs recognize their target sites and which binding events lead to mRNA repression. This is due to the reliance on indirect methods to infer these features, such as conservation of sites, mRNA expression level changes upon miRNA induction, and AGO binding sites via UV-crosslinking. Since these methods interrogate naturally occurring miRNA sites, they are most informative for more common sites and have less statistical power to address less common sites. Biochemical studies have only been performed on a few target sequences and suggest that RNA?RNA base pairing of miRNA-target interactions are greatly modified in the context of AGO, and consequently, simple base-pairing rules used in repression models are not predictive of AGO?miRNA binding affinity. Thus, the central hypothesis of this proposal is that in vitro binding affinity of the AGO?miRNA complex to mRNA targets sites is an unaccounted feature that will be predictive of changes in mRNA levels containing a particular site. This hypothesis is derived from the observed relationship between repression levels and the extent of complementarity of common miRNA-site types to their miRNA (i.e. repression of an 8mer > 7mer > 6mer). Therefore, the objective of this proposal is to determine the extent to which binding energetics of miRNA-AGO target complex account for the repression levels observed in vivo. First, human AGO-miRNA binding kinetics and thermodynamics will be determined using a high-throughput binding assay on pools of sequence variants of miRNA target sites. These data will allow determination of the contributions of miRNA?target sequence complementarity, structural accessibility, and primary sequence identity to binding affinity. These data will also provide insight into less common miRNA-site types whose functions are debated and largely unexplored. Principles derived from this analysis can be used to engineer AGO family proteins and synthetic miRNAs. Second, intracellular repression data will be generated for thousands of different sites and obtained by sequencing the levels of a reporter gene 3? UTR containing a library of miRNA binding-site variants. These repression data will include less common miRNA-site types and will be free from the effects of varying 3? UTR context. The binding rate and equilibrium constants acquired in vitro will be compared to the newly generated in vivo repression data to determine the extent to which binding affinity accounts for repression. These comparisons will provide insight into the features of miRNA-target recognition that are important for repression, and can be applied to the design of miRNA-based therapeutics.